Various animal studies have shown beneficial effects of hypercapnia in lung injury. However, in patients with acute respiratory distress syndrome (ARDS), there is controversial information regarding the effect of hypercapnia on outcomes. The duration of carbon dioxide inhalation may be the key to the protective effect of hypercapnia. We investigated the effect of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced lung injury in mice. C57BL/6 mice were randomly divided into a control group or an LPS group. Each LPS group received intratracheal LPS (2 mg/kg); the LPS groups were exposed to hypercapnia (5% carbon dioxide) for 10 min or 60 min before LPS. Bronchoalveolar lavage fluid (BALF) and lung tissues were collected to evaluate the degree of lung injury. LPS significantly increased the ratio of lung weight to body weight; concentrations of BALF protein, tumor necrosis factor-α, and CXCL2; protein carbonyls; neutrophil infiltration; and lung injury score. LPS induced the degradation of the inhibitor of nuclear factor-κB-α (IκB-α) and nuclear translocation of NF-κB. LPS increased the surface protein expression of toll-like receptor 4 (TLR4). Pre-treatment with inhaled carbon dioxide for 10 min, but not for 60 min, inhibited LPS-induced pulmonary edema, inflammation, oxidative stress, lung injury, and TLR4 surface expression, and, accordingly, reduced NF-κB signaling. In summary, our data demonstrated that pre-treatment with 10-min carbon dioxide inhalation can ameliorate LPS-induced lung injury. The protective effect may be associated with down-regulation of the surface expression of TLR4 in the lungs.
Keywords: acute respiratory distress syndrome; carbon dioxide; lipopolysaccharide; toll-like receptor 4
Acute respiratory distress syndrome (ARDS) develops most commonly in the context of severe sepsis, particularly when caused by infection with Gram-negative bacilli such as Escherichia coli [[
Hypercapnia and hypercapnic acidosis (HCA) exerts multiple important effects in lung injury and acute respiratory failure, which may be beneficial or deleterious to multiple biological pathways [[
However, HCA is not without risks. The safety of HCA in the setting of a live bacterial infection, such as pneumonia, remains a significant concern. Long-term exposure to HCA may impair the host response to an invading pathogen, permit bacterial proliferation, and ultimately worsen lung injury [[
To determine whether inhaled carbon dioxide had an anti-inflammatory effect in the lungs, mice were exposed to 5% carbon dioxide for 10 min or 60 min before LPS treatment (Figure 1). We evaluated the effect of inhaled carbon dioxide on the maintenance of body weight in LPS-induced lung injury in mice. Ten minutes, but not 60 min, of inhaled carbon dioxide attenuated the loss of body weight in LPS-induced lung injury in mice (Figure 2A). Intratracheal instillation of LPS caused lung injury, including pulmonary edema, microvascular protein leakage, and inflammatory cell infiltration. This lung injury was reflected in an increased ratio of lung weight to body weight (Figure 2B), in bronchoalveolar lavage fluid (BALF) protein concentration (Figure 2C), BALF lactate dehydrogenase (LDH) activity (Figure 2D), and BALF total cell count (Figure 2E). Carbon dioxide pre-treatment for 10 min, but not for 60 min, reduced the amount of pulmonary edema, microvascular protein leakage, cell damage, and inflammatory cell infiltration in the lungs considerably in the LPS-treated mice (Figure 2B–E). To examine whether pre-treatment with inhaled carbon dioxide could limit LPS-induced oxidative stress, the concentration of protein carbonyl in serum was measured by ELISA. Pre-treatment with inhaled carbon dioxide for 10 min or 60 min significantly reduced the concentration of protein carbonyl in serum from LPS-treated mice (Figure 2F).
LPS-induced production of TNF-α, a proinflammatory cytokine, chemokine CXCL2, and IL-10, an anti-inflammatory cytokine, in serum at 2 h (Figure 3A–C), in serum at 24 h (Figure 3D–F), in BALF at 2 h (Figure 3G–I), and in BALF at 24 h (Figure 3J–L). Both TNF-α and CXCL2, in the serum and BALF, at 2 and 24 h, were suppressed significantly by pre-treatment with inhaled carbon dioxide for 10 min, but not for 60 min. The shorter pre-treatment with inhaled carbon dioxide also substantially reduced the alveolar TNF-α (Figure 3J) and CXCL2 (Figure 3K) secretion at 24 h. Although TNF-α (Figure 3D) and CXCL2 (Figure 3E) in the serum at 24 h were suppressed by pre-treatment with inhaled carbon dioxide for 60 min, the concentration of CXCL2 (Figure 3H) in BALF at 2 h was significantly increased by pre-treatment with inhaled carbon dioxide for 60 min. Interesting, the concentration of IL-10, induced by LPS, in serum at 2 h was not suppressed by pre-treatment with inhaled carbon dioxide for 10 min, but was increased by pre-treatment with inhaled carbon dioxide for 60 min.
Increased inflammatory responses were confirmed by microscopic examination, which showed septal thickening, alveolar edema, and increased inflammatory cell infiltration 24 h after LPS (Figure 4A). Ten minutes of pre-treatment with inhaled carbon dioxide reduced the lung injury score (Figure 4B) and LPS-induced neutrophil sequestration in the lungs considerably (Figure 4C). These results are consistent with the immunohistochemistry staining for myeloperoxidase (MPO) (Figure 4D). The overall appearance of congestion and edema 24 h after LPS was improved by pre-treatment with inhaled carbon dioxide for 10 min (Figure 5).
The LPS-induced activation of the TLR4 and its downstream NF-κB signaling were investigated to determine the principles causing the anti-inflammatory actions of inhaled carbon dioxide. Pre-treatment with inhaled carbon dioxide for 10 min significantly inhibited LPS-induced IκBα degradation (Figure 6A) and NF-κB p65 nuclear translocation (Figure 6B) at 2 h.
To investigate the molecular mechanisms underlying the ability of inhaled carbon dioxide pre-treatment to attenuate LPS-induced lung injury, the expression of TLR4 protein in the lungs was assessed by Western blot analysis. The expression of TLR4 protein was maintained at 2 h (Figure 7A), but higher at 24 h (Figure 7B) after LPS administration. Pre-treatment with inhaled carbon dioxide for 10 min and for 60 min reduced TLR4 protein expression at 2 h after LPS stimulation. However, only pre-treatment with inhaled carbon dioxide for 10 min, but not for 60 min, significantly reduced LPS-induced TLR4 protein expression at 24 h after LPS stimulation (Figure 7B). The results were confirmed by immunohistochemical staining of TLR4 (Figure 7C). LPS increased but inhaled carbon dioxide pre-treatment suppressed TLR4 expression. The data suggest that the protective effects of inhaled carbon dioxide pre-treatment may be via suppression of TLR4 surface expression.
A schematic diagram of the second part of the experimental protocol is shown in Figure 8. To investigate the effects of inhaled carbon dioxide on TLR4 surface expression, the expression of TLR4 protein in the lungs was assessed by Western blot analysis (Figure 8). The TLR4 protein surface expression was significantly reduced after exposure to inhaled carbon dioxide for 10 min (Group 2) and 60 min (Group 4). Interestingly, suppression of TLR4 surface expression by the 10-min inhaled carbon dioxide was sustained for at least 1 h (Group 3). However, the level of TLR4 protein surface expression after exposure to inhaled carbon dioxide for 60 min (Group 4) was higher than the other two groups that exposed to inhaled carbon dioxide for 10 min (Group 2) and inhaled carbon dioxide for 10 min with room air for 50 min (Group 3).
We characterized the beneficial effects of inhaled carbon dioxide in a mouse model of LPS-induced lung injury. Instillation of LPS provoked leucocyte infiltration and cytokine production, NF-κB pathway activation, and increased alveolar capillary protein permeability, which suggests inflammatory lung injury. Pre-treatment with inhaled carbon dioxide for 10 min, but not 60 min, impeded NF-κB activation substantially, limited inflammatory cell invasion, and renewed the alveolar capillary integrity. These novel results suggest that pre-treatment with inhaled carbon dioxide for 10 min diminished LPS-induced lung damage by inhibiting the inflammatory reaction in the lungs.
These findings are consistent with those of other studies using a similar treatment protocol that revealed protective effects of inhaled carbon dioxide in animal models of LPS-induced lung injury [[
Previous studies have demonstrated the fundamental mechanisms by which HCA exerts beneficial effects, including attenuation of free radical production [[
Duration of exposure to HCA is critical for the application of inhaled carbon dioxide to prevent or limit lung injury. In bacterial pneumonia-related lung injury, the primary concern of HCA is impaired host innate immunity [[
Because of a possible dual role for TLRs in airway diseases, caution is needed when designing pulmonary TLR-based therapies [[
Short-term inhalation of carbon dioxide has been applied in many clinical situations. Previous studies have shown that inhaled carbon dioxide is efficacious in eliminating Cheyne–Stokes respiration with central sleep apnea [[
C57BL/6 adult male mice (8–10 weeks of age) were purchased from Charles River Technology in Taipei, Taiwan. All study animals were cared for in accordance with National Institutes of Health Guidelines (National Academy Press, Washington, DC, 1996).
The Institutional Animal Care and Use Committee at National Defense Medical Center and the National Science Council in Taipei, Taiwan approved the study (IACUC-14-101, 1 May 2014). The mice were subjected to carbon dioxide at ambient laboratory temperature in a large chamber fitted with automated controllers for carbon dioxide and oxygen. All mice were anesthetized during the procedures with intraperitoneal zolazepam–tiletamine (25 mg/kg of body weight; Zoletil
E. coli-derived lipopolysaccharide (LPS) (E. coli serotype 0111:B4, Sigma Chemical Company, St. Louis, MO, USA) was prepared at a concentration of 1 mg/mL in phosphate-buffered saline (PBS). A MicroSprayer aerosolizer (IA-1C; Penn-Century Inc., Philadelphia, PA, USA) inserted into the trachea delivered the LPS solutions into the lungs of the mice.
The mice were randomly assigned to the experimental groups. The experiment was divided into two parts. The first part of experiment included following 4 groups at 2 h (n = 6 in each group) and 24 h (n = 6 in each group) after PBS or LPS spray. Control (CON) group (Group 1): Intratracheal PBS (50 μL) was sprayed into the lungs of the mice in the control group. LPS group (Group 2): Same concentration of LPS (2 mg/kg) was sprayed into the lungs of the mice in the various experimental groups. Two of the LPS groups also received carbon dioxide by exposure to 5% carbon dioxide for 10 min (Group 3: H10L group) or 60 min (Group 4: H60L group) before the LPS spray. The primary outcome was the severity of lung damage. The secondary outcomes were the levels of inflammatory cytokines and oxidative stress markers, the loss of body weight, and the infiltrations of inflammatory cells. To investigate the effect of inhaled carbon dioxide without LPS on TLR4 expression in the lungs, the second part of experiment included following 4 groups (n = 6 in each group). CON group (Group 1) received room air for 60 min. H10 group (Group 2) received room air for 50 min followed by exposure to 5% carbon dioxide for 10 min. H10+50 group (Group 3) received carbon dioxide by exposure to 5% carbon dioxide for 10 min followed by room air for 50 min. H60 group (Group 4) received carbon dioxide by exposure to 5% carbon dioxide for 60 min.
At the end of the experiment, BALF was collected. The chest was surgically opened, and the trachea was exposed. An intravenous infusion needle was inserted. After ligating the hilum of the right lung, the left lung was lavaged by irrigating it with two separate 0.5-mL aliquots of PBS. One BALF aliquot was used immediately to measure the total cell count, and another BALF aliquot was used for measurement of lactate dehydrogenase (LDH) activity after centrifugation at 200× g for 10 min. LDH activity in BALF was assessed as described previously [[
Differences in serum protein carbonyl content were determined using a commercial kit (Carbonyl Protein Assay Kit, Cayman Chemical Company, Ann Arbor, MI, USA). Tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand 2 (CXCL2), and Interleukin-10 (IL-10) in the serum or BALF were assessed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems Inc., Minneapolis, MN, USA).
The mouse lung tissue was fixed in 10% neutral-buffered formalin for 24 h and then embedded in paraffin. The lung tissue samples were then sliced into 4-μm sections and stained with hematoxylin and eosin. Then, the severity of lung damage was scored as described previously [[
The paraffin-embedded mouse lung samples underwent immunohistochemical staining as described previously with minor modifications [[
Immunoblotting was performed as described previously [[
All data are expressed as means ± standard deviations (SDs). Differences were evaluated using Student's t-test, one-way analysis of variance, or two-way analysis of variance followed by Bonferroni's post-hoc test where appropriate. Differences were considered significant at a p-value <0.05. All calculations were made using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).
In conclusion, we demonstrated that pre-treatment with inhaled carbon dioxide for 10 min, but not for 60 min, reduced LPS-induced lung inflammation considerably in mice. This protective action may be associated with down-regulation of TLR4 surface expression. The prophylactic application and optimal treatment timing for carbon dioxide inhalation require additional investigation.
Graph: Figure 1 Schematic diagram of the first part of experimental protocol. The experiment was divided into two parts. The first part of the experiment included following 4 groups at 2 and 24 h (n = 6 in each group) after phosphate-buffered saline (PBS) or lipopolysaccharide (LPS) spray. Control (CON) group (Group 1): Intratracheal PBS (50 μL) was sprayed into the lungs of the mice in the CON group. LPS group (Group 2): Same concentration of LPS (2 mg/kg) was sprayed into the lungs of the mice in the various experimental groups. Two of the LPS groups also received carbon dioxide by exposure to 5% carbon dioxide for 10 min (Group 3: H10L group) or 60 min (Group 4: H60L group) before the LPS spray.
Graph: Figure 2 The effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced weight loss, pulmonary edema, inflammatory lung injury, inflammatory cell infiltration, and oxidative stress. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. (A) Body weight loss was recorded 24 h after LPS administration (n = 9 per group). (B) The lung weight to body weight ratio (LW/BW) was recorded 24 h after treatment. Total protein concentration (C) and lactate dehydrogenase (LDH) activity (D) were measured in bronchoalveolar lavage fluid (BALF) collected 24 h after treatment. (E) Total cell count was determined in BALF. (F) The concentration of protein carbonyl in serum 24 h after LPS administration was measured by ELISA. The data are expressed as the mean ± standard deviation (SD) (n = 6 per group). * Significantly different from the CON group (* p < 0.05, ** p < 0.01, *** p < 0.001). + Significantly different from the LPS group (+p < 0.05, ++p < 0.01, +++p < 0.001). # Significantly different from the H10L group (#p < 0.05).
Graph: Figure 3 The effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced changes in TNF-α, CXCL2, and IL-10 concentrations. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. The concentrations of TNF-α (A), CXCL2 (B), and IL-10 (C) in the serum at 2 h; TNF-α (D), CXCL2 (E), and IL-10 (F) in the serum at 24 h; TNF-α (G), CXCL2 (H), and IL-10 (I) in the bronchoalveolar lavage fluid (BALF) at 2 h; and TNF-α (J), CXCL2 (K), and IL-10 (L) in the BALF at 24 h after LPS administration were measured by enzyme-linked immunosorbent assay (ELISA). The data are expressed as the mean ± SD (n = 6 per group). * Significantly different from the CON group (* p < 0.05, ** p < 0.01, *** p < 0.001). + Significantly different from the LPS group (+p < 0.05, ++p < 0.01, +++p < 0.001). # Significantly different from the H10L group (#p < 0.05, ##p < 0.01, ###p < 0.001).
Graph: Figure 4 The effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced lung injury and inflammatory cell infiltration at 24 h. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. (A) Representative images of hematoxylin and eosin-stained lung sections from one of the six mice per experimental group are presented. Original magnification = 40×. (B) The lung injury score and (C) neutrophil count were obtained from the images. The data are expressed as the mean ± SD. * p < 0.05, *** p < 0.001, compared with the CON group; +++p < 0.001, compared with the LPS group; ##p < 0.01, ###p < 0.001, compared with the H10L group. (D) Representative images of myeloperoxidase (MPO)-stained lung sections from one of the six mice per experimental group are presented. Original magnification = 40×.
Graph: Figure 5 Gross lung pathology to demonstrate the effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced lung injury. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. Representative images from one of the six mice per experimental group are presented.
Graph: Figure 6 The effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced NF-κB activation. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. The cytosolic levels of IκBα (A) and nuclear levels of NF-κB p65 (B) in lung tissues 2 h after LPS administration were analyzed by Western blot analysis and are expressed as fold change relative to α-tubulin and TATA, respectively. The data are expressed as the mean ± SD. * Significantly different from the CON group (p < 0.05). + Significantly different from the LPS group (p < 0.05). # Significantly different from the H10L group (p < 0.05).
Graph: Figure 7 The effects of pre-treatment with inhaled carbon dioxide on lipopolysaccharide (LPS)-induced toll-like receptor 4 (TLR4) expression. C57BL/6 mice were challenged with LPS (2 mg/kg intratracheally) with or without pre-treatment with inhaled carbon dioxide for 10 min (H10L) or 60 min (H60L) before LPS administration. Control (CON) animals were treated with PBS solution. The surface expression of TLR4 was analyzed from the membrane fractions of lung tissues at 2 h (A) and 24 h (B) after LPS by Western blot analysis and is expressed as fold change relative to α-tubulin. The data are expressed as the mean ± SD. * Significantly different from the CON group (p < 0.05). + Significantly different from the LPS group (p < 0.05). # Significantly different from the H10L group (p < 0.05). (C) Representative images of TLR4-stained lung sections from one of the six mice per experimental group are presented. Original magnification = 40×.
Graph: Figure 8 Schematic diagram of the second part of experimental protocol and the effects of carbon dioxide inhalation on toll-like receptor 4 (TLR4) expression. To investigate the effect of inhaled carbon dioxide on TLR4 expression in the lungs, the second part of experiment included following 4 groups at 1 h (n = 6 in each group). Control (CON) group (Group 1): C57BL/6 mice were treated with room air for 60 min. H10 group (Group 2): Mice were treated with room air for 50 min followed by inhaled carbon dioxide for 10 min. H10+50 group (Group 3): Mice were treated with inhaled carbon dioxide for 10 min followed by room air for 50 min. H60 group (Group 4): Mice were treated with inhaled carbon dioxide for 60 min. The surface expression of TLR4 was analyzed in the membrane fractions of lung tissues at the indicated time points by Western blot analysis and is expressed as fold change relative to α-tubulin. The data are expressed as the mean ± SD. * Significantly different from the CON group (** p < 0.01, *** p < 0.001). + Significantly different from the H10 group (p < 0.05). # Significantly different from the H10+50 group (p < 0.05).
Conceptualization, S.-E.T., S.-Y.W., C.-P.W., and K.-L.H.; methodology, S.-J.C., Y.-S.T., C.-K.P., and C.-C.L.; writing—original draft preparation, S.-E.T.; writing—review and editing, K.-L.H. and C.-P.W.; supervision, W.-C.P.
This study was supported, in part, by grants MOST 106-2314-B-016-036-MY3 from the Ministry of Science and Technology, Taiwan, TSGH-C105-088 and TSGH-C106-071 from Tri-Service General Hospital, and MAB-105-053, MAB-106-063, MAB-106-064, and MAB-107-085 from the National Defense Medical Center, Taiwan.
The authors declare no conflicts of interest.
By Shih-En Tang; Shu-Yu Wu; Shi-Jye Chu; Yuan-Sheng Tzeng; Chung-Kan Peng; Chou-Chin Lan; Wann-Cherng Perng; Chin-Pyng Wu and Kun-Lun Huang
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